Dihydroactinospectacin, amides and esters thereof



United States Patent 3,165,533 DIHYDROACTINOSPECTACIN, AMIDES AND ESTERSTHEREOF Herman Hoelrsema, Kalamazoo, and Paul F. Wiley, Texas Township,Kalamazoo County, Mich., assignors to The 5 Upjohn Company, Kalamazoo,Mich., a corporation of Delaware No Drawing. Filed Jan. 22, 1962, Ser.No. 167,931 7 Claims. (Cl. 260340.3)

This invention is related to novel compositions of mat- 10 ter and to aprocess for the preparation thereof, and is particularly directed to thenovel compound, dihydroactinospectacin, and its acyl derivatives and toa process for producing the same.

Actinospectacin is a biosynthetic product produced by the controlledfermentation of Streplomyces spectabilis. Various methods for theproduction, recovery, and purification of actinospectacin are describedin the published literature, e.g., D. J. Mason et al., Antibiotics andChemotherapy, 11 p. 118, 1961; M. E. Bergy et al., Antibiotics andChemotherapy, 11, p. 661, 1961; Union of South Africa Patent No. 60/4098and Belgian Patent No. 596,- 175. Actinospectacin is characterized by anoptical rotation [a] -20 (H O); by solubility in water, methanol, andethanol, and by insolubility in acetone and hydrocarbon solvents; and bythe presence of two basic groups: pKa, 6.95 (H O), pKa 8.70 (H 0). Itwas formerly thought that the molecular formula was C H N O- but it hassince been shown that the formula is C14H24N2O7.

It has now been found that a novel compound, according to thisinvention, is obtained by the reduction of actinospectacin. Onreduction, actinospectacin is converted to dihydroactinospectacin whichupon acylation is converted to its di-N-acyl and tetra-N,O-acylderivatives. 30

Subsequent to this invention, the structure of actinospectacin has beenelucidated. Dihydroactinospectacin,

therefore, can now be represented by the following formula:

O O CH l I 1o 1 cm 7 3 B0 6 3/ 4 IYTCH; H H OH On acylation with twomoles of acylating agent, there is obtainedN,N'-diacyldihydroactinospectacin which on further acylation yieldsN,N'-diacyldihydroactinospectacin 4,9-diacylate having the followingformula:

| 0 0 can: 8 9 1o 1 2 CH3 NC I H: OR R 0H rated or unsaturated, straightor branched chain aliphatic carboxylic acid, for example, acetic,propionic, butyric, isobutyric, tert-butylacetic, valeric, isovaleric,caproic, caprylic, decanoic, dodecanoic, acrylic, crotonic, hexynoic,heptynoic, octynoic acids, and the like; (b) a saturated or unsaturatedalicyclic carboxylic acid, for example, cyclobutanecarboxylic acid,cyclopentanecarboxylic acid, cyclopentenecarboxylic acid,methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid,dimethylcyclohexenecarboxylic acid, dipropylcyclohexanecarboxylic acid,and the like; (0) a saturated or unsaturated substituted alicyclicaliphatic carboxylic acid, for example, cyclopentaneacetic acid,cyclopentanepropionic acid, cyclopenteneacetic acid, cyclohexanebutyricacid, methylcyclohexaneacetic acid, and the like; (d) an aromaticcarboxylic acid, for example, benzoic acid, toluic acid, naphthoic acid,ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, andthe like; and (e) an aromatic-aliphatic carboxylic acid, for example,phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamicacid, phcnylpropiolic acid, naphthylacetic acid, and the like.

The term halogen-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, andlower alkoxy-substituted hydrocarbon carboxylic acid acyl'of from two totwelve carbon atoms, inclusive is intended to mean hydrocarboncarboxylic acid acyl as hereinbefore defined which are substituted byone or more halogen atoms, nitro, hydroxy, amino, cyano, thiocyano, orlower alkoxy groups. By lower alkoxy is meant an alkoxy group of fromone to six carbon atoms, inclusive, for example, methoxy, ethoxy,propoxy, butoxy, pentyloxy, hexyloxy, and isomeric forms thereof.Examples of substituted hydrocarbon carboxylic acid acyl falling withinthe above definition are the acyl corresponding to chloroacetic acid,chloropropionic acid, bromobutyric acid, iodovaleric acid,chlorocyclohexanecarboxylic acid, o-, m-, and p-chlorobenzoic acid,anisic acid, salicylic acid, p-hydroxybenzoic acid, 0-, m-, andp-nitrobenzoic acid, cyanoacetic acid, thiocyanoacetic acid,cyanopropionic acid, lactic acid, glycine, ethoxyformic, and the like.

Dihydroactinospectacin is obtained either by chemical or catalyticreduction of actinospectacin. In either case the 0x0 group in theactinospectaciu molecule is reduced to an oxy group. Thus, on reactingactinospectacin with sodium borohydride, or on hydrogenation ofactinospectacin with platinum oxide, dihydroactinospectacin is obtained.In both cases the dihydroactinospectacin is usually recovered as a saltwhich can be converted to the free base by treatment with a stronglybasic anion exchange resin. [Suitable anion exchange resins for thispurpose are obtained by chloromethylating by the procedure given onpages 88 and 97 of Kunin, Ion Exchange Resins, 2nd Ed., (1958), JohnWiley and Sons, Inc., polystyrene cross-linked, if desired, withdivinylbenzene prepared by the procedure given on page 84 of Kunin,supra, and quaternizing with trimethylamine or dimethylalkanolamine bythe procedure given on page 97 of Kunin, supra. Anion exchange resins ofthis type are marketed under the trade names Dowex-2, Dowex-20,Amberlite IRA-400, Duolite A-102, and Permutit S-l. Crudedihydroactinospectacin can be upgraded by passage over a cation exchangeresin. Both carboxylic acid and sulfonic acid types can be used.Suitable carboxylic acid resins include the polyacrylic acid resinsobtained by the copolymerization of acrylic acid and divinylbenzene bythe procedure given on page 87 of Kunin, Ion Exchange Resins, 2nd ed.,(1958), John Wiley and Sons, Inc. Carboxylic acid cation exchange resinsof this type are marketed under the trade names Amberlite IRC-SO and 0Zeokarb 226. Suitable sulfonic acid resins include nuclear sulfonatedpolystyrene resins crosslinked with divinylbenzene obtained by theprocedure given on page 84 of Kunin, supra. Sulfonated cation exchangeresins of this type are marketed under the trade names Dowex-SO,Amberlite IR-120, Nalcite HCR, Chempro C-ZO, Permutit Q, and Zeokarb225.]

The di-N-acyl and tetra-N,O-acyl derivatives of dihydroactinospectacin,according to the invention, are obtained by acylatingdihydroactinospectacin. When dihydroactinospectacin is reacted with anappropriate acid anhydride or chloride, the corresponding N,N-diacylandN,N-diacyldihydroactinospectacin 4,9 -diacylate are obtained. Forexample, on reacting dihydroactinospectacin with about two moles ofacetic anhydride in the presence of pyridine,N,N'-diacetyldihydroactinospectacin is obtained. On further acylation ofthe diacyl or on acylation of actinospectacin with four or more moles ofacetic anhydride, N,N-di-acetyldihydroactinospectacin 4,9-diacetate isobtained. Also, on reacting dihydroactinospectacin with ethylchloroformate (ethyl chlorocarbonate) in the presence of pyridine,N,N'-diethoxycarbonyldihydroactinospectacin andN,N'-diethoxycarbonyldihydroactinospectacin 4,9-bis-(ethyl carbonate)are obtained according to the proportions of acylating agent used. Mixedacylates can be obtained by acylating theN,N'-diacyldihydroactinospectacin with a different acylating agent. Forexample, on reacting N,N'-diacetyldihydroacetinospectacin with sodiumpropionate and propionic anhydride the corresponding mixed acylate isobtained.

Various salts of dihydroactinospectacin can be made by contacting thestarting material with acids, for example, hydrogen chloride, hydrogenbromide, hydrogen iodide, sulfuric acid, phosphoric acid, picric acid,helianthic acid, Reineckes acid, succinic acid, maleic acid, latic acid,trichloroacetic acid, and the like. These salts are useful asintermediates from which high purity dihydroactinospectacin and its acylderivatives can be recovered when desired by treatment with an anionexchange resin as described above.

The novel compound of the invention, dihydroactinospectacin, hasantibacterial activity as shown in the following Table I.

TABLE I Microorganism: MIC* (meg/ml.) Escherichia coli 32 Proteusvulgaris 250 Klebsiella pneumoniae 16 Pseudomonas aeruginosa 250Salmonella typhosa 64 Staphylococcus aureus 32 Staphylococcus albus 64Streptococcus jaecalis 32 Minimum inhibitory concentration.

The assay was run using a standard streptomycin assay broth at a pH of8.0, (beef extract 1.5 gm./liter, yeast extract 3.0 gm./liter, peptone 6gm./liter in distilled water and adjusted to pH 8.0 aftersterilization).

Dihydroactinospectacin is useful in preventing the growth andmultiplication of various microorganisms in many environments. It can beused, for example, to de- Iiquefaciens which causes infectious dropsy orto treat fish affected by this organism. Also, this compound can be usedto inhibit the growth of the bacterium, Flavobacterium suaveolans whichis a common contaminant of the papermill industry. Further, this novelcompound can be used to inhibit the growth of Erysipelothrixrhusiopathiae which is a pathogen found in swine, sheep, turkeys,pigeons, and mice.

The novel compounds of the invention, N,N-diacyldihydroactinospectacinand N,N'-diacyldihydroactinospectacin 4,9-diacylate are useful asintermediates to make 1,3- deoxy-l,3-bis(methylamino)myoinositol whichhas been given the trivial name actinamine. Actinarnine can beacetylated to hexaacetylactinamine. The reaction can be carried out byacid hydrolysis of the starting material, e.g.,N,N'-diacyldihydroactinospectacin or N,N'-diacyldihydroactinospectacin4,9-diacylate. Hydrolysis is effected by contact thereof with an acidicmaterial, e.g., a strong mineral acid such as hydrogen chloride,hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, andthe like. Preferably, hydrochloric acid is used as a hydrolyzing agentand when so used the actinamine is isolated as the hydrochloride. Theother acids listed may also be used for the hydrolysis and when so usedthe product is isolated as the corresponding mineral acid salt. The

actinamine can then be further acylated to hexaacetylactinamine.Actinarnine is useful jn accordance with US. Patents 1,915,334 and2,075,359, in preparing amine fiuosilicatemoth-p'r'oofing agents, and inaccordance with U.S."Pat'e nts 2,425,320 and 2,606,155, in preparingamine thiocyanate-formaldehyde condensation products for use as picklinginhibitors. Hexaacetylactinamine has antibacteiial activity and can beused to decontaminate aquaria of the fish pathogen Aeromonasliquefaciens which causes infectious dropsy or to treat fish affected bythis organism. Also, this novel compound can be used to inhibit thegrowth of the bacterium Flavobacterium suaveolans which is a commoncontaminant of the papermill industry. Further, this novel compound canbe used to inhibit the growth of Erysipelothrix rhusiopathiae which is apathogen found in swine, sheep, turkeys, pigeons, and mice.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting.

Example I.-Dihydroactinospectacin A. Catalytic reduction: A suspensionof 2.7 g. of actinospectacin dihydrochloride in ml. of 50% ethanol washydrogenated in the presence of 300 mg. of platinum oxide for 3 days at40 p.s.i.g. hydrogen pressure in a Parr apparatus. Following filtrationand evaporation to an aqueous solution, acetone was added andcrystallization occurred, yielding 1.45 g. of dihydroactinospectacindihydrochloride with a melting point of 203-210" C. Recrystallizationyielded dihydroactinospectacin dihydrochloride crystals having a meltingpoint of 205208 C. and an optical rotation [a] =+33 (C., 1% in water).

Elemental analysis.-Calculated for C, 41.28; H, 6.93; C], 17.41. Found:C, 41.78; H, 7.12; Cl, 17.39.

B. Borohydr-ide reduction: Sodium borohydride (2.3 g.) was added slowlyto a solution of 10 g. of actinospectacin in 500 ml. of anhydrousmethanol. After standing at room temperature for 2 /2 hrs. the solutionwas adjusted to pH 3.0 with 6.0 N hydrochloric acid, and then evaporatedto dryness under reduced pressure. The resulting residue was dissolvedin methanol, filtered, and the filtrate evaporated to dryness underreduced pressure. This was done twice using 250 ml. portions ofmethanol, once using ml. of methanol, and once using 50 ml. of methanol.The final residue was dissolved again in 50 ml. of methanol andprecipitated with ether. The precipitate was removed by filtration,dried in a vacuum desiccator and then dissolved in 3.0 N hydrochloricacid using 4 ml. of acid per gram of precipitate. Upon the addition ofacetone to this solution and refrigerating, crystallization occurred,yielding 3.8 g. of material melting at 199 to 205 C. Two grams of thismaterial was dissolved in 20 ml. of water and passed over 30 g. of astrongly basic anion exchange resin (the anion exchange resin used forthis purpose was obtained by chloromethylating by the procedure given onpages 88 and 97 of Kunin, Ion Exchange Resins, 2nd Ed., (1958), JohnWiley and Sons, Inc., polystyrene crosslinked with 5% divinylbenzeneprepared by the procedure given on page 84 of Kunin, supra, andquaternized with trimethylamine by the procedure given on page 97 ofKunin, supra). The column was washed with water until the washings were5 neutral. The combined washings and emuent were freeze-dried. Theresidue was then dissolved in water and put on 30 g. of a carboxylicacid cation exchange resin. (The carboxylic acid cation exchange resinused for this purpose was obtained by the copolymerization of acrylicacid and 5% divinylbenzlene by the procedure given on page 87 of Kunin,Ion Exchange Resins, 2nd Ed., (1958), John Wiley and Sons, Inc.) Thecolumn was eluted with 300 ml. of 1.0 N hydrochloric acid. The

' eluate was adjusted to pH 4.0 with a strongly basic anion exchangeresin of the type described above. The resin was removed and thesolution freewdried and then recrystallized twice from a water-acetonemixture to yield dihydroactinospectacin dihydrochloride crystals havinga melting point of 204-206 C., a pKa of 7.0 and a pKaof 8.8, anequivalent weight of 211.5, and an optical rotation [a] :-28 (c.==1% inwater).

Elemental analysis-Calculated for C I-1 N 2HC1: C, 41.28; H, 6.93; N,6.88; C], 17.41. Found: C, 43.54; H, 7.15; N, 6.57; Cl, 17.05.

The dihydrochloride was converted to the base by the followingprocedure. A solution of 2 g. of dihydroactinospectacin dihydrochloridein 20 ml. of water was poured over 30 g. of a strongly basic anionexchange resin of the type previously described. The column was washedwith water until the washings were neutral. The combined efiiuent andwashings were freeze-dried to yield 1.3 g. of dihydroactinospectacin.

Elemental analysis-Calculated for C H N O C, 50.39; H, 7.84; N, 8.38.Found: C, 49.59; H, 8.24; N, 8.24

Example 2.--N,N-Diacety ldihydroactinospeclacz'n 20.3 g. ofdihydroactinospectacin base was dried by vacuum distilling 200 ml. ofabsolute ethanol from it. This was repeated. The drydihydroactinospectacin base was then dissolved in 600 ml. of pyridineand the solution evaporated in vacuo to 100 ml. To this was slowly added9.45 ml. of acetic anhydride with stirring and chilling. The solutionwas then stored 3 days at room temperature. Volatile products wereremoved by vacuum distillation and the residue was dissolved in 50 ml.of pyridine and 100 ml. of chloroform. Precipitation occurred on theaddition of 4 to 6 vols. of Skellysolve B (isomeric hexanes) to yield 23g. of product. This material was then fractionated in a Craigcountercurrent distribution apparatus using a solvent system n-butanol:water (1:1) to yield 13.8 g. of N,N'-diacetyldihydroactinospectacinhaving an optical rotation [a] =15 (c., 1% in ethanol). Potentiometrictitration indicated the absence of any basic functions.

Elemental analysis.-Calculated for C H N O z C, 51.66; H, 7.23; N, 6.70;C-CH 10.8. Found: C, 50.73; H, 7.72; N, 6.67; C-CH 10.9.

Example 3.N,N-Diacetyldihydroactinospectacin 4,9-Diacetare A solution of10 g. of dihydroactinospectacin base and 300 ml. of pyridine wasdistilled to 100 ml. in vacuo. The resulting solution was diluted to 200ml. with dry pyridine and treated with 5.8 ml. of acetic anhydride.After eight days of storage at room temperature, the readily volatileproducts were removed by distillation in vacuo, and the residue wasleached with ethyl acetate to remove 900 mgs. ofN,N'-diacetyldihydroactinospectacin. The remaining residue wasredissolved in 100 ml. of pyridine and treated with ml. of aceticanhydride. After three days the volatile materials were removed asbefore, and the residue was dissolved in ethyl acetate and precipitatedwith Skellysolve B. This precipitate (6.65 g.) was distributed in acountercurrent distribution using water and n-butanol (1:1) as thesystem for 500 transfers. N,N-diacetyldihydroactinospectacin4,9-diacetate was isolated from the peak fractions as a white amorphoussolid with a melting point of 175 to 6 C. and an optical rotation [a] =8(c., 1% in chloroform).

Elemental analysis.Ca1culated for C H N O C, 52.58; H, 6.82; N, 5.58;acetyl, 33.50 (molecular weight, 502.51). Found: C, 52.39; H, 6.87; N,5.37; acetyl, 32.80.

Example 4 By substituting the acetic anhydride in Example 2 by propionicanhydride, there is obtained N,N-dipropionyldihydroactinospectacin.

Example 5 By substituting the acetic anhydride in Example 3 by propionicanhydride, there is obtained N,N-dipropionyldihydroactinospectacin4,9-diacetate.

Example 6 By substituting the acetic anhydride and pyridine in Example 2by acetyl chloride, propionyl bromide, butyryl chloride, valerylchloride, caproyl chloride, heptanoyl chloride, and caprylyl chloride,and at least a stoichiometric amount of triethylamine there are obtainedN,N- diacetyl-, N,N-dipropionyl-, N,N-dibutyryl-, N,N'-divaleryl-,N,N-dicapr0l-, N,N-diheptanoyl-, and N,N-dicaprylyldihydroactinospectacin.

Example 7 By substituting the acetic anhydride and pyridine in Example 3by acetyl chloride, propionyl bromide, butyryl chloride, valerylchloride, caproyl chloride, heptanoyl chloride, and caprylyl chloride,and at least a stoichiometric amount of triethylamine there are obtainedN,N- diacetyldihydroactinospectacin 4,9-diacetate,N,N-dipropionyldihydroactinospectacin 4,9-dipropionate,N,N-dibutyryldihydroactinospectacin 4,9-dibutyrate,N,N'-divaleryldihydroactinospectacin 4,9-divalerate,N,N'-dicaproyldihydroactinospectacin 4,9-dicaproate,N,N'-diheptanoyldihydroactinospectacin 4,9-diheptanoate, andN,N'-dicaprylyldihydroactinospectacin 4,9-dicapry1ate.

Example 8 By substituting the acetic anhydride and pyridine in Example2, by methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octylchlorocarbonate and at least a stoichiometric amount of triethylaminethere are obtained N,N- dimethoxycarbonyldihydroactinospectacin, N,N'diethoxycarbonyldihydroactinospectacin, N,N'dipropoxycarbonyldihydroactinospectacin, N,Ndibutoxycarbonyldihydroactinospectacin, N,N'dipentyloxycarbonyldihydroactinospectacin, N,N'dihexyloxycarbonyldihydroactinospectacin, N,N'diheptyloxycarbonyldihydroactinospectacin, andN,N'-dioctyloxycarbonyldihydroactinospectacin.

Example 9 7 8 We claim: 5. N,N'-diacetyldihydroactinospeclacin. 1. Acompound selected from the class consisting of 6. A compound of theformula: dihydroactinospectacin of the formula:

B OH O O 5 cn r z- 9 1o 1 om s s cn,N- 9 1o 1 on,

2 HO 7 5 3 S S 6 \0/ 4 H0 5 3 O IIICH; R f H 10 R OH H OH wherein R andR are alkanoyl of from 2 to 12 carbon atoms, inclusive.

and the acid addition salts thereof' 7.N,N'-diacetyldihydroactinospectacin 4,9-diacetate.

2. Dihydroactinospectacin.

3. Dihydroactinospectacin dihydrochloride. 15 References Cit d b theExaminer 4. A compound of the formula. UNITED STATES PATENTS R 0H1,964,973 7/34 Bockmiihl et a1 260-3403 O 0 2,975,193 3/61 Dice et al260326.5 -8 S 3 2,997,471 8/61 Cheney et a1 260-559 H0 7 5/ \/8 OTHERREFERENCES 0 I Bergy et al.: Antibiotics and Chemotherapy," vol. 11,RICH; 0H page 662 (1961). R H IRVING MARCUS, Primary Examiner.

wherein R is alkanoyl of from 2 to 12 carbon atoms, D, T, McCUTCHEN,WALTER A, MODANCE, inclusive. Examiners.

1. A COMPOUND SELECTED FROM THE CLASS CONSISTING OFDIHYDROACTINOSPECTACIN OF THE FORMULA: